Growing evidence suggests the possible presence of rare and highly tumorigenic progenitor cells within breast tumors that may be major contributors to cancer development, progression, metastasis, and resistance to therapy. Cell-surface marker analysis and serial transplantations in appropriate hosts have indicated that a majority of this "breast tumor-initiating activity" resides in the population that overexpresses CD44 (hyaluronan receptor) among other surface characteristics. Hyaluronan (HA) is an extracellular matrix polysaccharide that has two functionally linked cellular receptors: CD44 and RHAMM. Both receptors are linked to breast tumor invasion and progression. Preliminary data from a model of a highly tumorigenic human breast cancer cell line that resembles breast tumor progenitor subpopulations suggest these cells have a higher rate of HA-probe internalization than do non-progenitor cell lines. The hypothesis to be tested is that these minor populations will also overexpress RHAMM protein that results in a high rate of HA-probe internalization. A long-term goal is to target the tumorigenic progenitor cells at single-cell resolution. Thus, the proposed research seeks to integrate life and physical sciences/techniques to accurately profile the putative tumorigenic progenitor markers and finely demarcate these minorities within breast tumor cells. Specific Aim I. To screen for surface display of putative tumorigenic progenitor markers and to assess HAprobe sensitivity and uptake mechanism(s). We will use fluorescent activated cell sorting (FACS) for multiplex profiling of surface markers and will assess HA-probe uptake sensitivity and specificity through genetic over-expression and fluorescent microscopy. In order to identify the role of RHAMM and/or CD44 receptors in rapid uptake of HA-probe and internalization mechanism (s,) we will use inhibitory antibody and/or knockdown experiments. Specific Aim II. To demarcate highly positive subpopulations that uptake HA-probes rapidly and to a higher content than do the rest of the cells. We will use cell-patterned platforms, FACS and confocal fluorescent microscopy for fine demarcation and high -content quantization of internalization events. Specific Aim III. To validate the progenitor capability of the highly tumorigenic populations that rapidly internalize HA-probes. We will use FACS, serial transplantation in mice and 3D cultures to test the tumorigenicity and progenitor-like characteristics of sorted fractions based on selective surface markers. Upon establishment of proof of principle, cells from primary human breast tumors are obtained to test for the generality of the approach. Successful targeting of minor tumor subsets may establish the basis for high-resolution simultaneous molecular targeting and therapy for breast tumor, enable improved resection of such tumors, and increase the therapeutic index of available anti-tumor compounds.